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The effects of the non-dialyzable products of pepsin digested serum proteins on the growth of tissue cells in vitro

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The effects of the non-dialyzable products of pepsin digested serum proteins on the growth of tissue cells in vitro

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Content THE EFFECTS OF THE NON-DIALYZABLE PRODUCTS OF
PEPSIN DIGESTED SERUM PROTEINS ON THE
GROWTH OF TISSUE CELLS i n v i t r o
A T h e s is
P r e s e n t e d t o
The F a c u l t y o f th e D e p a rtm e n t o f B io c h e m is tr y an d N u t r i t i o n
The U n i v e r s i t y o f S o u th e rn C a l i f o r n i a
I n P a r t i a l F u l f i l l m e n t
o f t h e R e q u ire m e n ts f o r t h e D egree
M a s te r o f S c ie n c e
b y
A l b e r t D. W illia m s
Ju n e 1949
This thesis, written by
--ALBEET-B.-HILLIAMS.......................
under the guidance of h% 3.... Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
_.mST.ER....QP....S.CJ;ENGE...................
E. S. B ogardus,
........................................................ ©isarr.........................
Date .........................
Faculty Committee
TABUS OP CONTENTS
CHAPTER PAGE
I INTRODUCTION........................ 1
I I HISTORICAL REVIEW ....................................................................... 3
I I I ROUTINE CULTURE TECHNIQUES .................................................. 8
A. P r e p a r a t i o n o f b i o l o g i c a l m a t e r i a l s . . . 8
B . P r e p a r a t i o n o f s o l u t i o n s .......................................... 9
C. T re a tm e n t o f e q u i p m e n t ................................................ 11
D* The c u l t u r e room • 11
E . P ro c e d u re f o r s e t t i n g up c u l t u r e s . . . . 12
IV EXPERIMENTAL METHODS................................................................. 14
A. P r e p a r a t i o n o f serum d i g e s t s ................................. 14
B . C h em ical m eth o d s .................................... • • • . . 18
C. F i r s t e x p e r i m e n t a l s e r i e s . . . . . . . . 25
1 . P r e l i m i n a r y t r e a t m e n t ................................ 25
2 . E x p e r im e n ta l c u l t u r e s ........................... 25
3 . C o n tr o l c u l t u r e s ................................................ 25
D. Second e x p e r i m e n t a l s e r i e s .................................... 27
1 . P r e l i m i n a r y t r e a t m e n t ................................. 27
2 . E x p e r im e n ta l c u l t u r e s . . . . . . . 28
3 . C o n tr o l c u l t u r e s . . . . • • . • • • 28
4 . S u b s e q u e n t t r e a t m e n t . • • • . . . • 28
iv
CHAPTER PAGE
V EXPERIMENTAL D A T A .......................................................................... 30
A. Serum d i g e s t s ....................................................... 30
B* P i r s t e x p e r i m e n t a l s e r i e s ...................................... 30
C* S econd e x p e r i m e n t a l s e r i e s ........................ 36
VI DISCUSSION OP R E S U L T S ................................................................ 46
A. E f f e c t s o f p e p s i n on serum p r o t e i n s . . . 46
B. E f f e c t o f d i g e s t on g ro w th o f c u l t u r e s . • 47
V I I SUMMARY............................................. 51
*
BIB LIO G R A PH Y ....................................................................... 52
LIST OP TABLES
TABLE PAGE
I . C o m p o sitio n o f Simms ,!X7M P h y s i o l o g i c S o l u t i o n 10
I I . D i g e s t i o n M ix tu re s ..... • 14
I I I . T e m p e ra tu re V a r i a t i o n in t h e B u f f e r D u rin g
D i g e s t i o n • • • • • . . . . . 19
IV . D a ta f o r C a l i b r a t i o n C urve ........................................ 21
V. Amount o f Serum D ig e s t and Simms S o l u t i o n Used
i n E ach E x p e r im e n ta l C u l t u r e ................................. . 26
V I. R e s u l t s o f P e p s in D i g e s t io n o f Serum P r o t e i n s 31
V I I . D e c re a s e in T o t a l N itr o g e n an d NPN D u rin g
D i g e s t io n an d D i a l y s i s •• .• • • • • • • • 32
V I I I . R e s u l t s o f th e F i r s t E x p e r im e n ta l S e r i e s . . . 33
IX . P l a n i m e t e r M easurem ents o f C u l t u r e s i n t h e
S econd E x p e r im e n ta l S e r i e s .............................. 37
X. A v erag e P l a n im e te r R e a d in g s o f C u l tu r e s I n E ac h
D ig e s t o f th e S econd E x p e r im e n ta l S e r i e s • • 38
X I . P e r C ent I n c r e a s e In A rea o f C u l t u r e s in th e
S econd E x p e r im e n ta l S e r i e s ................................... 38
LIST OP FIGURES
FIGURE PAGE
I , A ssem bled D i g e s t i o n an d D i a l y s i s A p p a ra tu s . . . 16
I I . D e t a i l s o f D i g e s t i o n and D i a l y s i s A p p a ra tu s . • 17
I I I . C a l i b r a t i o n C urve f o r N itr o g e n D e te r m in a tio n s 23
IV . C u rv es Show ing P e r C ent I n c r e a s e i n A rea o f
C u l tu r e s Grown i n Serum D i g e s t s : A (pH 2 . 0 ) ,
B (pH 2 . 5 ) , C (pH 3 .0 ) an d D (pH 3 .5 ) . . . 39
V. M ic ro p h o to g ra p h s a t 24 Hour I n t e r v a l s o f T y p ic a l
C u l tu r e s Grown i n E ach Serum D i g e s t :
(a ) D i g e s t s A (pH 2 .0 ) an d B (pH 2 .5 )
fro m 0 t o 76 h o u r s ........................................ 40
(b ) D i g e s t s A (pH 2 .0 ) and B (pH 2 .5 )
fro m 96 t o 144 h o u rs . . . . . . . . 41
(c ) D i g e s t s C (pH 3 .0 ) and D (pH 3 .5 )
fro m 0 t o 76 h o u rs ................................... 42
(d ) D i g e s t s C (pH 3 . 0 ) and D (pH 3 .5 )
fro m 96 t o 144 h o u r s ..................................... 43
CHAPTER I
INTRODUCTION
I t h a s b e e n o b s e rv e d t h a t e n z y m a tic d i g e s t i o n o f
serum p r o t e i n s p ro d u c e s s u b s ta n c e s w h ic h h a v e g ro w th s tim u
l a t i n g a c t i v i t y f o r t i s s u e c e l l s jLn v i t r o * A lso known i s
t h e f a c t t h a t t h e m o le c u la r w e ig h ts o f p o l y p e p t i d e s p ro d u c e d
by an e n z y m a tic h y d r o l y s i s o f p r o t e i n w i l l v a r y a c c o r d i n g t o
t h e pH and t h e te m p e r a tu r e a t w h ic h th e d i g e s t i o n i s p e r
fo rm e d . The p r e s e n t i n v e s t i g a t i o n was c a r r i e d o u t t o o b t a i n
e v id e n c e w h ic h w ould i n d i c a t e w h e th e r o r n o t p o l y p e p t i d e s
can b e u t i l i z e d a s fu n d a m e n ta l u n i t s f o r th e s y n t h e s i s o f
p r o t e i n b y t i s s u e c e l l s g ro w in g i n v i t r o .
A m ethod b y w h ic h a s im u lta n e o u s e n z y m a tic d i g e s t i o n
an d d i a l y s i s o f serum p r o t e i n s c o u ld be p e rfo rm e d u n d e r
a s e p t i c c o n d i t i o n s h a s b een u t i l i z e d i n t h i s i n v e s t i g a t i o n .
C h e m ica l a n a l y s e s o f th e d i g e s t s h a v e b e e n u s e d t o i n d i c a t e
th e e x t e n t o f e n z y m a tic h y d r o l y s i s .
To e v a l u a t e th e r e l a t i v e g ro w th s t i m u l a t i n g a c t i v i t y
o f t h e v a r i o u s d i g e s t s u s e d , m ic ro p h o to g r a p h s o f t h e e n t i r e
c e l l c o lo n y a t r e g u l a r tim e i n t e r v a l s , h a v e .been m ade. I n a s
much a s a n i n c r e a s e in a r e a o f th e c u l t u r e i s n o t a t r u e
c r i t e r i a o f a c t u a l g ro w th b u t may p a r t i a l l y b e a r e s u l t o f
c e l l m i g r a t i o n , m ic ro p h o to g ra p h s a t h i g h e r m a g n i f i c a t i o n s
hav e b e e n made t o d e m o n s tra te t h e c h a r a c t e r i s t i c s o f th e
c e l l s a s g ro w th p r o g r e s s e s * M ic ro s c o p ic o b s e r v a t i o n s o f
t h e c u l t u r e s a t f r e q u e n t i n t e r v a l s h a v e b e e n made t o e s
t i m a t e t h e h e a l t h o f t h e c e l l s i n t h e c u l t u r e s *
C u l t u r e s o f c h ic k e n em bryo h e a r t t i s s u e h av e b e e n
s e l e c t e d f o r u s e in t h i s e x p e rim e n t s i n c e t h i s ty p e o f
t i s s u e i s c o n v e n i e n t l y grow n i n C a r r e l f l a s k c u l t u r e s .
The r e s p o n s e o f th e c e l l s t o t h e a d d i t i o n o f t h e d i g e s t s
can b e o b s e rv e d in s h o r t p e r i o d s o f tim e and t h e e x t e n t
4
o f th e a c t i v i t y o f t h e d i g e s t s i n te rm s o f i n c r e a s e i n a r e a
o f t h e c u l t u r e s can b e a p p ro x im a te d b y th e m ic r o p h o to g r a p h i
te c h n iq u e w i t h l i t t l e d i f f i c u l t y *
CHAPTER I I
HISTORICAL REVIEW
B urrow s and Neymann (1 917) d e m o n s tr a te d t h a t t i s s u e
c e l l s c u l t i v a t e d in h a n g in g d ro p c u l t u r e s w i t h m edia con
s i s t i n g o f a v a r i e t y o f am ino a c i d m ix t u r e s f a i l e d t o grow ,
and d e v e lo p e d a t o x i c a p p e a ra n c e * C a r r e l an d h i s c o -w o rk e rs
(19 26) r e p e a t e d some o f t h e e x p e r im e n ts o f B urrow s a n d Neymann
w i t h m o d i f i c a t i o n s i n te c h n iq u e a n d o b t a i n e d s u b s t a n t i a l l y
t h e same r e s u l t s * C om paring t h e e f f e c t s o f d i a l y z e d and n o n -
d i a l y z e d em bryo e x t r a c t on c e l l g ro w th , C a r r e l o b s e rv e d o n ly
a s l i g h t g ro w th s t i m u l a t i n g a c t i v i t y o f th e d i a l y z e d em bryo
e x t r a c t * S in c e th e em bryo e x t r a c t h a d b e e n d i a l y z e d f r e e o f
am ino a c i d s and no s t i m u l a t i o n o f g r o w th was o b s e rv e d when
am ino a c i d m ix t u r e s w ere a d d e d , C a r r e l c o n c lu d e d t h a t c e l l s
in v i t r o w ere u n a b le t o u t i l i z e am ino a c i d n i t r o g e n f o r p r o
t e i n s y n t h e s i s *
L a t e r B a k e r an d C a r r e l (1 9 2 8 a) d e m o n s tr a te d t h e g ro w th
s t i m u l a t i n g a c t i v i t y o f p r o t e o s e s o b t a i n e d b y e n z y m a tic
d i g e s t i o n o f p r o t e i n s . The g e n e r a l f e e l i n g a t t h a t tim e was
t h a t t i s s u e c e l l s g ro w in g i n v i t r o w ere u n a b le t o u t i l i z e
am ino a c i d s f o r t h e s y n t h e s i s o f p r o t e i n b u t r a t h e r r e q u i r e d
some h i g h e r c le a v a g e p r o d u c t s o f t h e p r o t e i n m o le c u le a s a
s o u rc e o f n i t r o g e n f o r p r o t e i n s y n t h e s i s b y t h e c e l l *
4
However a s F i s c h e r (1 9 4 7 ) i n d i c a t e d i n h i s re v ie w
o f p r o t e i n m e ta b o lis m o f t i s s u e c e l l s g ro w in g i n v i t r o ,
t h e e a r l i e r w o rk e rs o v e rlo o k e d a s i g n i f i c a n t f a c t *
One com ponent o f t h e m e d ia w h ic h was u s e d b y t h e s e
w o rk e rs c o n s i s t e d o f p la sm a w h ic h c o n ta in e d n o rm a l c o n c e n
t r a t i o n s o f am ino a c i d s * The f a i l u r e o f th e c e l l s t o grow
i n t h e d i a l y z e d em bryo e x t r a c t i n C a r r e l 's e x p e r im e n ts was
d u e , n o t t o th e re m o v a l o f am ino a c i d s , b u t r a t h e r t o t h e
e l i m i n a t i o n o f some o t h e r d i a l y z a b l e g ro w th f a c t o r * The
t o x i c e f f e c t s o b s e rv e d when am ino a c i d s w ere a d d e d t o t h e
c u l t u r e s w ere no d o u b t due t o h ig h c o n c e n t r a t i o n s r e s u l t i n g
when t h e am ino a c i d s w ere a d d e d t o t i s s u e s i n p la s m a con
t a i n i n g s u f f i c i e n t am ounts o f t h e am ino a c i d s f o r n o rm al
grow th* M o re r a p i d g ro w th s h o u ld n o t have b e e n a n t i c i p a t e d
a s a r e s u l t o f th e a d d i t i o n o f am ino a c i d m ix t u r e s t o t i s s u e
c u l t u r e s g ro w in g i n m ed ia c o n t a i n i n g t h e n e c e s s a r y c o n c e n
t r a t i o n s o f am ino a c i d s *
T o x ic e f f e c t s on t h e s u b s t r a t e hav e b e e n n o te d i n t h e
l i t e r a t u r e f r e q u e n t l y when a s u b s ta n c e was a d d ed i n am ounts
w h ic h e x c e e d e d t h e r e q u i r e d c o n c e n t r a t i o n f o r a n o rm a l r e
sp o n se *
F i s c h e r (1941) ren ew ed t h e i n v e s t i g a t i o n o f p r o t e i n
m e ta b o lis m o f t i s s u e c e l l s g ro w in g i n v i t r o , u t i l i z i n g a
m o d i f i c a t i o n in te c h n iq u e * B o th d i a l y z e d serum a n d d i a l y z e d
em bryo e x t r a c t w ere p r e p a r e d . A d d itio n o f t h i s m ix tu r e t o
c u l t u r e s o f h e a l t h y c e l l s g ro w in g i n C a r r e l f l a s k s p ro d u c e d
a m arked e f f e c t r e s u l t i n g i n c e l l u l a r d e g e n e r a t i o n an d a
r a p i d c e s s a t i o n o f g r o w th . A lso no g ro w th o c c u r r e d when
f r e s h t i s s u e e x p l a n t s w ere p l a c e d i n t h e c o m p le te ly d i a l y z e d
m e d ia . In a d d i t i o n t o t h e s e r e s u l t s , F i s c h e r (1 9 4 2 a) was
a l s o a b le t o d e m o n s tr a te t h a t th e a d d i t i o n o f c e r t a i n am ino
a c i d m i x t u r e s t o t h e d i a l y z e d m ed ia su p p le m e n te d th e g ro w th
s t i m u l a t i n g a c t i v i t y o f t h e m e d ia . G row th s t i m u l a t i n g a c t i
v i t y was r e s t o r e d t o th e e x t e n t t h a t g ro w th o f t i s s u e c e l l s
was o b s e r v e d , a lt h o u g h l e s s r a p i d l y th a n i n n o rm al c o n t r o l
c u l t u r e s i n n o n - d i a l y z e d em bryo e x t r a c t an d se ru m .
The e f f e c t o f h i g h e r c le a v a g e p r o d u c t s o f p r o t e i n s
on t h e g ro w th o f t i s s u e c e l l s was s t u d i e d b y F i s c h e r and
A s tru p (1942) an d d i s c u s s e d b y F i s c h e r ( 1 9 4 6 ) . E n z y m a tic
d i g e s t s o f b o t h hom ologous and h e t e r o l o g o u s seru m w ere p r e
p a r e d . T h e se d i g e s t s w ere a d d ed t o t i s s u e c e l l s g ro w in g
i n v i t r o an d t h e f o l lo w in g i n t e r e s t i n g o b s e r v a t i o n s w ere made
A d d itio n o f p e p s i n d i g e s t e d hom ologous seru m t o t i s s u e c e l l s
in an o th e r w is e c o m p le te ly d i a l y z e d medium p ro d u c e d a g ro w th
r e s p o n s e w h ic h was c o m p a rab le t o t h e g ro w th o b s e rv e d in th e
p o s i t i v e c o n t r o l c u l t u r e s g ro w in g i n n o n - d i a l y z e d em bryo
e x t r a c t a n d se ru m . I n a d d i t i o n , t h e m o rp h o lo g y o f t h e c e l l s
in t h e e x p e r i m e n t a l c u l t u r e s was n o rm a l.
More c o m p le te d e g r a d a t i o n o f serum p r o t e i n s b y t h e
a c t i o n o f t r y p s i n p ro d u c e d d i g e s t s w h ic h i n c r e a s e d th e tim e
6
r e q u i r e d f o r t h e i n i t i a t i o n o f grow th* The r a t e o f 'g r o w t h
w as l e s s t h a n t h a t o b s e rv e d i n c o n t r o l c u l t u r e s *
C o m p lim en tary t o t h e s e o b s e r v a t i o n s was a n o t h e r e x
p e r im e n t i n w h ic h F i s c h e r a d d ed a p e p s i n d i g e s t o f h e t e r o l
ogous serum t o t i s s u e c e l l s jtn v i t r o * In t h i s c a s e , g ro w th
was p o o r a n d n o t u n t i l s m a l l e r c le a v a g e p r o d u c t s o b t a i n e d
b y d i g e s t i n g h e te r o lo g o u s seru m w i t h t r y p s i n w ere a d d ed t o
t h e c u l t u r e s d id g ro w th o c c u r . I n a d d i t i o n , t h e m o rp h o lo g y
o f t h e c e l l s i n t h e t r y p s i n - d i g e s t e d h e t e r o l o g o u s serum was
a t y p i c a l *
The p o s s i b i l i t y o f a s p e c i f i c s t r u c t u r a l c o n f i g u r a
t i o n o f th e m o le c u le a s a p r e r e q u i s i t e t o t h e i n c o r p o r a t i o n
o f p o l y p e p t i d e s i n t o t i s s u e c e l l s was p o s t u l a t e d b y F i s c h e r
( 1 9 4 7 ), b a s e d upon t h e r e s u l t s o f h i s work*
The p o s s i b i l i t y e x i s t s t h a t p e p s i n d i g e s t s o f hom ol
ogous p r o t e i n s p ro d u c e d p o l y p e p t i d e s w i t h a s t r u c t u r a l
c o n f i g u r a t i o n w h ic h p e r m i t t e d t h e m o le c u le t o b e u s e d i n t a c t
f o r t h e s y n t h e s i s o f p r o t e i n * No f u r t h e r e n z y m a tic d e g r a d a
t i o n was n e c e s s a r y . When p e p s i n - d i g e s t e d h e te r o l o g o u s
serum was u s e d , t h e c o n f i g u r a t i o n o f t h e p o l y p e p t i d e s
p ro d u c e d w ould n o t p e r m it t h e i r d i r e c t u t i l i z a t i o n b y t h e c e l l
f o r p r o t e i n s y n t h e s i s * $ o t u n t i l f u r t h e r d e g r a d a t i o n b y
t r y p s i n was a c c o m p lis h e d c o u ld t h e s p l i t p r o d u c t s b e d i r e c t
l y u s e d b y t h e c e l l s .
D e u ts c h (1946 ) an d a l s o B ridg em an (1 9 4 6 ) d e m o n s tr a te d
t h a t t h e l e n g t h s o f p o l y p e p t i d e s o b t a i n e d b y e n z y m a tic
d i g e s t i o n v a r i e s b o t h w i t h th e pH and t h e t e m p e r a t u r e a t
w h ic h t h e d i g e s t i o n i s p e rf o r m e d . A maximum y i e l d o f 6 0 -7 0
p e r c e n t o f h a l f m o le c u le s o f g l o b u l i n ( d e te r m in e d e l e e t r o -
p h o r e t i c a l l y ) was o b t a i n e d b y B ridgem an a t a pH o f 3 .5 an d
t e m p e r a t u r e o f 1- 2° G.
CHAPTER I I I
ROUTINE CULTURE TECHNIQUES
A. PREPARATION OP BIOLOGICAL MATERIALS
E x t r a c t fro m t e n d a y o l d New H am pshire Red c h ic k
em bryos was u s e d a s t h e n o rm al g ro w th s t i m u l a n t . T h is e x
t r a c t , s t o r e d in s t e r i l e v i a l s , was f r o z e n i n a d r y i c e
c h e s t t o d e s t r o y an y c e l l s w h ic h may h a v e b e e n p r e s e n t . The
m ethod o f f r e e z i n g a l s o r e d u c e d b a c t e r i a l c o n ta m i n a ti o n .
The m a t e r i a l was wanned t o 37 ° C. b e f o r e u s e . P lasm a was
o b t a i n e d b y a s e p t i c c a n n u l a t i o n o f th e c a r o t i d a r t e r i e s o f
a d u l t New H am p sh ire Red c h i c k e n s . The b lo o d was c o l l e c t e d
i n c h i l l e d , s t e r i l i z e d c e n t r i f u g e t u b e s w h ic h h a d b e e n
c o a te d w i t h a l a y e r o f ' ^ r i f l l m ’* t o a v o id c lo ttin g .-* - R a p id
c e n t r i f u g a t i o n was f o llo w e d b y re m o v a l o f t h e s u p e r n a t a n t
p la sm a w h ic h was s t o r e d in d r i f l l m e d s t e r i l e v i a l s i n t h e
r e f r i g e r a t o r . When serum was n e e d e d , p la sm a was t r a n s f e r r e d
fro m th e v i a l s t o s t e r i l e b o t t l e s an d was p e r m i t t e d t o c l o t
i n t h e i n c u b a t o r . The c l o t was c o m p le te ly d i s i n t i g r a t e d by
p r o lo n g e d s h a k in g w ith s t e r i l e g l a s s b e a d s and w ashed s a n d .
F o llo w in g r a p i d c e n t r i f u g a t i o n , t h e s u p e r n a t a n t serum was
rem oved a n d s t o r e d i n t h e r e f r i g e r a t o r .
1 ’'D r i f i l m ” i s a p r o d u c t p ro d u c e d b y G e n e r a l E l e c t r i c
C o r p o r a t i o n . C o l l e c t i n g t u b e s a r e c o a te d w i t h t h i s s u b s ta n c e ,
an d c o a g u l a t i o n o f b lo o d i s d e la y e d .
B. PREPARATION OP SOLUTIONS
9
Simms s o l u t i o n , a p h y s i o l o g i c a l s o l u t i o n u s e d f o r
w a sh in g t i s s u e f r a g m e n ts and c u l t u r e s , was p r e p a r e d a c c o r d
in g t o d i r e c t i o n s g iv e n i n T a b le I# Simms s o l u t i o n was a l s o
u s e d t o d i l u t e em bryo e x t r a c t f o r u s e I n th e c u l t u r e s . The
pH o f th e s o l u t i o n was a d j u s t e d tO',7.4 an d s t e r i l i z e d b y
S e i t z f i l t r a t i o n i n t o a s t e r i l e c o n t a i n e r . S t e r i l e 25 m l.
b o t t l e s w ere f i l l e d w i t h t h e f i l t e r e d Simms s o l u t i o n , cap p ed
w i t h s t e r i l e s l e e v e s t o p p e r s an d l e f t i n t h e I n c u b a t o r a t
3 9 ° C. f o r 72 h o u rs t o d e t e c t c o n ta m in a tio n I f p r e s e n t . The
b o t t l e s , I f fo u n d t o b e s t e r i l e , w ere s t o r e d i n th e r e f r i g
e r a t o r .
To a i d In d e te r m i n i n g th e pH o f t h e t i s s u e c u l t u r e ,
p h e n o l r e d i n d i c a t o r was u s e d a s d e s c r i b e d b y P a r k e r (1 9 3 8 ) .
One h u n d re d m il l ig r a m s o f p h e n o l r e d w ere g ro u n d w i t h 2 .8 2
m l. o f 0 .1 0 N NaOH u n t i l d i s s o l v e d . The s o l u t i o n was r i n s e d
i n t o a 100 m l. v o l u m e t r i c f l a s k and d i l u t e d t o volum e w ith
d i s t i l l e d w a t e r . The r e s u l t i n g s o l u t i o n c o n t a i n i n g 0 . 1 p e r
c e n t p h e n o l r e d was d i s p e n s e d i n s m a ll b o t t l e s , and s t e r i l
i z e d In t h e a u t o c l a v e . P h o s p h a te b u f f e r s w e re p r e p a r e d t o
g iv e s o l u t i o n s o f fro m pH 7 .0 t o pH 7 . 8 . T hese w ere s t e r i l i z e d
b y a u t o c l a v i n g .
The i n d i c a t o r i n 0 .0 5 m l. am ounts was a d d e d t o 1 m l.
o f e a c h o f t h e s t e r i l e b u f f e r s c o n ta in e d i n s t e r i l i z e d C a r r e l
f l a s k s . The f i n a l c o n c e n t r a t i o n o f p h e n o l r e d In t h e f l a s k
x o
TABLE I
COMPOSITION OP SIMMS "XV" PHYSIOLOGIC SOLUTION2
(IN GRAMS PER LITER)
S u b s ta n c e Amount
NaCl . . . * ....................................
KC1 ................................................................
C aC l2 *2H20 . . . . . ................. . .
MgCl2 - 6 H 2 0 ............................... ...................................
NaHC03 . ....................... . . . . . .
NagjHPO^ •••••••••••••••
D e x tro s e • • • • • • • • • • • • • • •
2 P r e p a r e d M edia f o r T is s u e C u l t u r e
b i o l o g i c a l A s s o c i a t e s I n c . , F le m in g to n , New
(1948) M ic ro -
J e r s e y .
IX
was 0 .0 0 5 p e r c e n t . T h is c o n c e n t r a t i o n h a s b e e n r e p o r t e d b y
P a r k e r (1938) t o b e n o n - t o x i c t o t i s s u e c e l l s . When c u l t u r e s
w e re s e t u p , 0 .0 5 m l. o f t h e I n d i c a t o r , m e a su re d w i t h a 1 m l.
t u b e r c u l i n s y r i n g e was a d d e d f o r e a c h 1 m l. o f m ed ia i n th e
c u l t u r e . The c o l o r o f t h e m ed ia i n t h e c u l t u r e c o u ld th e n
b e com pared v i s u a l l y w i t h t h e b u f f e r s t a n d a r d s a n d t h e pH
a d j u s t e d t o t h e d e s i r e d ra n g e b y g a s s i n g w i t h CO2 p a s s e d
th r o u g h a s t e r i l e f i l t e r o r b y a r e a t i o n a s r e q u i r e d .
C. TREATMENT OP EQUIPMENT
A l l g la s s w a r e u s e d in t h e t i s s u e c u l t u r e e x p e r im e n ts
was so a k e d i n d ic h ro rn a te c l e a n i n g s o l u t i o n , th o r o u g h ly
w ashed i n r u n n in g t a p w a t e r and f i n a l l y r i n s e d i n t w i c e -
d i s t i l l e d w a t e r . A f t e r d r y i n g i n t h e h o t a i r o v e n , a l l
tu b e s a n d f l a s k s w ere p lu g g e d w ith c o t t o n , w rap p ed and
s t e r i l i z e d f o r t h r e e h o u r s a t 1 5 0 ° C. I n s t r u m e n t s w ere
w ashed i n so a p , r i n s e d , d r i e d an d s t e r i l i z e d in t h e same w ay.
D. THE CULTURE ROOM
The c u l t u r e room was e q u ip p e d w i t h s e v e r a l M S t e r i l a m p s ,,,
one o f w h ic h was l o c a t e d d i r e c t l y above th e c u l t u r e t a b l e .
A l l c u l t u r e w ork was c a r r i e d o u t' d i r e c t l y u n d e r t h i s u l t r a
v i o l e t lam p . V e n t i l a t i o n o f t h e c u l t u r e room was a c c o m p lis h e d
b y f o r c i n g a i r o v e r b a n k s o f ’IS t e r i l a m p s ,,. T e s ts w e re made
o f th e s t e r i l i t y o f th e a i r i n th e c u l t u r e room b y p l a c i n g
12
open P e t r i d i s h e s o f m ed ia a t v a r i o u s p l a c e s in t h e c u l t u r e
room . R e s u l t s fro m t h e s e t e s t s i n d i c a t e d t h a t t h e p o s s i
b i l i t y o f c o n ta m in a tio n o f t h e c i i l t u r e s b y b a c t e r i a and m olds
on d u s t p a r t i c l e s was n e g l i g i b l e .
E . PROCEDURE FOR SETTING UP CULTURES
C u l t u r e s o f c h ic k e n em b ry o n ic h e a r t t i s s u e h a v e b e e n
m a i n t a i n e d in C a r r e l f l a s k s u s i n g a m o d i f i c a t i o n o f a t e c h
n iq u e d e s c r i b e d b y P a r k e r (1 9 3 8 ).
F ra g m e n ts o f a p p r o x im a te ly one c u b ic m i l l i m e t e r
w ere s e c t i o n e d fro m t h e h e a r t o f a t e n d a y New H am pshire
Red c h ic k e n em bryo. A f t e r s e v e r a l w a s h in g s i n Simms
s o l u t i o n , one fr a g m e n t was p la c e d i n t h e c e n t e r o f e a c h
C a r r e l f l a s k c o n t a i n i n g 0 . 5 m l. o f a 1 :2 0 d i l u t i o n o f embryo
e x t r a c t i n Simms s o l u t i o n an d 0 .0 5 m l. o f p h e n o l r e d i n
d i c a t o r . O n e - h a lf o f a m i l l i l i t e r o f c h ic k p la sm a was
a d d ed t o t h e f l a s k a n d th e fra g m e n t was c e n t e r e d b e f o r e
c l o t t i n g o c c u r r e d . The pH was a d j u s t e d t o b e tw e e n 7 . 2 and
7 .6 b y g a s s i n g w i t h C0 2 » A f t e r a f i r m c l o t had fo rm ed , t h e
c u l t u r e s w ere in c u b a te d a t 39° C. P a t c h i n g w i t h m ore p la sm a
was done t w e n t y - f o u r h o u r s l a t e r when n e c e s s a r y .
T h e r e a f t e r t h e c u l t u r e s w ere w ashed a t f o r t y - e i g h t
h o u r i n t e r v a l s w i t h s e v e r a l 1 m l. p o r t i o n s o f Simms s o l u t i o n .
The w a s h in g s w ere a s p i r a t e d fro m th e f l a s k w i t h a s t e r i l e
n e e d l e . A f r e s h 1 :2 0 d i l u t i o n o f em bryo e x t r a c t in Simms
13
s o l u t i o n was p r e p a r e d . E q u a l v o lum es o f th e em bryo e x t r a c t
a n d c h ic k e n serum w ere p la c e d in t h e f l a s k s f o l lo w in g th e
l a s t w a sh in g w i t h Simms s o l u t i o n . I n d i c a t o r was a d d e d an d
t h e pH a d j u s t e d a s d e s c r i b e d p r e v i o u s l y .
A l l c u l t u r e s w ere rem oved fro m t h e i n c u b a t o r a n d
r e t u r n e d t o t h e i n c u b a t o r a t t h e same tim e so t h a t e a c h
c u l t u r e in a s e r i e s was e x p o se d t o t h e same t e m p e r a t u r e a t
a l l t i m e s ,
w The u s e o f h e p a r i n i z e d p la sm a was d i s c o n t i n u e d
s i n c e h e p a r i n was fo u n d t o b e t o x i c t o t h e t i s s u e c u l t u r e s
i n some c a s e s .
CHAPTER IV
EXPERIMENTAL METHODS
A. PREPARATION OP SERUM DIGESTS
A s t e r i l e s o l u t i o n o f r e c r y s t a l l i z e d p e p s i n i n
s a l i n e was p r e p a r e d c o n t a i n i n g 1 .1 0 0 rag. o f p e p s i n p e r m l.
o f s o l u t i o n . The volum e o f t h i s p e p s i n s o l u t i o n w h ic h was
u s e d f o r t h e d i g e s t i o n was c a l c u l a t e d so t h a t t h e same amount
o f p e p s i n p e r gram o f p r o t e i n i n th e serum t o h e d i g e s t e d
w as u s e d e a c h tim e , B ridgm an ( 1 9 4 6 ) . In T a h le I I a r e l i s t e d
t h e am oun ts o f serum a n d p e p s i n u s e d f o r e a c h d i g e s t . The
pH f o r e a c h d i g e s t i o n m ix tu r e i s a l s o i n d i c a t e d I n t h e t a b l e .
TABLE I I
DIGESTION MIXTURES
pH o f Volume in m i l l i l i t e r s
D i g e s t B u f f e r Serum P e p s in
A 2 .0 2 0 .0 1 .0 5
B 2 .5 2 5 .0 1 .3 0
C 3 .0 1 5 .0 0 .6 2
D 3 .5 2 5 .0 1 .0 0
15
T w e n ty - s ix l i t e r s o f a b u f f e r e d i s o t o n i c s o l u t i o n
w ere p r e p a r e d c o n t a i n i n g NaCl ( 8 .0 gm. p e r l i t e r ) and
NaH2P0 4 (2 .3 7 gm. p e r l i t e r ) . The p r o p e r pH f o r e a c h d i
g e s t i o n a s i n d i c a t e d i n T a b le I I (pag e 13) was o b t a i n e d b y
t h e a d d i t i o n o f s m a ll am ounts o f 12 N HC1.
C h ic k e n serum and p e p s in in th e am ounts d e f i n e d in
T a b le I I (p ag e 14) w ere p la c e d I n a c e llo p h a n e d i a l y z i n g
t u b e w h ic h h a d b e e n s t e r i l i z e d i n t h e a u t o c l a v e . The d i a l y z
in g tu b e was th e n su s p e n d e d I n 6 . 5 l i t e r s o f s t e r i l e b u f f e r
a t th e d e s i r e d pH an d p r e v i o u s l y c h i l l e d t o 1 -2 ° C. i n t h e
c o ld room . A Beckman pH m e te r w i t h e x t e n s i o n e l e c t r o d e s was
u s e d t o p e r i o d i c a l l y c h ec k th e pH o f t h e b u f f e r . A p p r o p r i a t e
c a l c u l a t i o n s w ere made to c o n v e r t a c t u a l pH r e a d i n g s on th e
i n s t r u m e n t t o t r u e pH v a lu e s o f t h e b u f f e r a t t h e t e m p e r a t u r e
o f t h e c o ld ro o m .^
The e n t i r e d i g e s t i o n and d i a l y z i n g a p p a r a t u s p i c
t u r e d in F i g u r e I an d F i g u r e I I was th e n p la c e d i n a l a r g e r
j a r c o n t a i n i n g w a te r t o a c t a s an i n s u l a t i n g b a t h w h ic h
p r e v e n t e d f l u c t u a t i o n s o f th e t e m p e r a t u r e in th e c o ld room
fro m b e i n g r e f l e c t e d In t h e b u f f e r . I n t h i s way t h e te m p e r
a t u r e o f t h e b u f f e r was m a in ta in e d a t a c o n s t a n t l e v e l .
4
Beckman B u l l e t i n 95-A , N a t io n a l T e c h n ic a l
L a b o r a t o r i e s , S o u th P a s a d e n a , C a l i f o r n i a .
FIGURE I
ASSEMBLED DIGESTION AND DIALYSIS APPARATUS
FIGURE I I
DETAILS OF DIGESTION AND DIALYSIS APPARATUS
18
T a b le I I I c o n t a i n i n g p h y s i c a l d a t a r e c o r d e d d u r in g
t h e p r e p a r a t i o n o f one o f th e d i g e s t s i s s u b m itte d t o i n
d i c a t e th e r e l a t i v e c o n s i s t a n c y o f t h e pH v a l u e s and th e
te m p e r a t u r e o f t h e b u f f e r .
A f t e r a 72 h o u r d i g e s t i o n an d d i a l y s i s p e r i o d , t h e
pH was a d j u s t e d t o 7 . 5 and th e d i a l y s i s b a g was t r a n s f e r r e d
t o a f l a s k c o n t a i n i n g f o u r l i t e r s o f s a l i n e . D i a l y s i s co n
t i n u e d i n t h i s s o l u t i o n f o r a n a d d i t i o n a l 72 h o u r s . A f t e r
t h e se c o n d p e r i o d o f d i a l y s i s , th e d l a l y z a b l e m a t e r i a l was
e s t i m a t e d t o have b e e n d i l u t e d 400 t i m e s . F o llo w in g t h e
d i a l y s i s , t h e serum w as rem oved a s e p t i c a l l y from t h e b a g ,
c e n t r i f u g e d an d t h e c l e a r s u p e r n a t a n t was s t o r e d i n th e
r e f r i g e r a t o r .
B . CHEMICAL METHODS
T o t a l n i t r o g e n and n i t r o g e n n o t p r e c i p i t a t e d b y 10
p e r c e n t t r i c h l o r a c e t i c a c i d ( h e r e a f t e r r e f e r r e d t o a s NPN)
w ere d e te r m in e d on e a c h sam ple o f serum b e f o r e a n d a f t e r t h e
e n z y m a tic d i g e s t i o n . A m o d if ie d m i c r o - K j e l d a h l t e c h n iq u e
was u s e d f o r t h e n i t r o g e n d i g e s t i o n w i t h d i r e c t n e s s l e r -
i z a t i o n o f an a l i q u o t . The y e llo w c o l o r w as r e a d i n a
Colem an s p e c t r o p h o t o m e t e r a t a wave l e n g t h o f 425 m i l l i
m ic r o n s .
A c a l i b r a t i o n c u rv e was p r e p a r e d u s i n g a s t a n d a r d
n i t r o g e n s o l u t i o n c o n t a i n i n g 6 .3 2 m ic ro g ra m s o f n i t r o g e n
19
TABLE I I I
TEMPERATURE VARIATION IN THE BUFFER DURING DIGESTION
D a te Hour
pH o f
B u f f e r
T e m p e ra tu re
C old
Room B u f f e r
F e b , 18 1 0 :1 5 AM 3 .5 3 4 ° F . 2 . 5 ° C.
ti
2 :3 0 PM
it
32 1 .5
tt
5 :3 0 PM
ti
3 2 ,5 1 .5
F e b . 19 1 2 :0 0 PM
it
33 1 .5
tt
3 :0 0 PM
ti
3 2 .5 1 .0
F e b • 21 7 :3 5 AM
ii
35 1 .5
n
1 0 :1 5 AM
tt
35 1 .5
20
p e r m i l l i l i t e r * T h re e in d e p e n d e n t sa m p le s w ere ru n f o r e a c h
c o n c e n t r a t i o n o f n i t r o g e n and t h r e e r e a d i n g s w ere made on
th e s p e c t r o p h o t o m e t e r , c h e c k in g th e b l a n k r e a d i n g e a c h
tim e * D a ta f o r th e c a l i b r a t i o n c u rv e a r e r e c o r d e d i n T a b le
IV .
A v erag e v a lu e s o f t h e p e r c e n t t r a n s m i s s i o n w ere
c a l c u l a t e d f o r e a c h c o n c e n t r a t i o n o f n i t r o g e n u s e d . T hese
v a lu e s w ere p l o t t e d on s e m ilo g a r lth m ic p a p e r and a s can b e
se e n i n F i g u r e I I I (p ag e 2 3 ) , a c u rv e was o b t a i n e d , th e
m a jo r p o r t i o n o f w h ic h was l i n e a r . I n t h e e x p e r im e n ts ,
unknown sa m p le s f o r t h e n i t r o g e n d e t e r m i n a t i o n , a f t e r a
p r e l i m i n a r y ru n t o e s t i m a t e th e a p p ro x im a te c o n c e n t r a t i o n ,
w ere d i l u t e d a p p r o p r i a t e l y t o o b t a i n a r e a d i n g w hich f e l l
b e tw e e n 20 and 50 p e r c e n t t r a n s m i s s i o n , th e l i n e a r p o r t i o n
o f th e c a l i b r a t i o n c u r v e .
The m i c r o - K j e l d a h l d i g e s t i o n s w ere done b y a m odi
f i e d m ethod d e s c r i b e d b y Wong ( 1 9 2 3 ) . A l l n i t r o g e n d e te r m in
a t i o n s w ere r u n in t r i p l i c a t e .
P r e l i m i n a r y t r e a t m e n t o f th e serum f o r t h e t o t a l
p r o t e i n d e t e r m i n a t i o n c o n s i s t e d o f p r e p a r i n g a 1 :5 0 d i l u t i o n
o f th e serum w i t h 0 .9 p e r c e n t s a l i n e . An a l i q u o t ( u s u a l l y
1 t o 3 m l .) o f t h e s o l u t i o n was d i g e s t e d w i t h 1 m l. o f a
1 :1 s o l u t i o n o f H2S0^ a n d w a t e r . When th e d i g e s t i o n had
beg un t o c l e a r , 0 .5 m l. o f a s a t u r a t e d p o ta s s iu m p e r s u l f a t e
TABLE- IV
DATA FOR CALIBRATION CURVE
t \
P er c e n t T r a n s m i s s i o n ^ )
S t a n d a r d '* ' 1 :5 0 H2SO4 M icrogram s Tube Number
S e r i e s
( in m l . )
( i n m l . ) o f N itro g e n
1 2 3 A verage
1 . 0 .0 5 .0 0 .0 100
1 1 .8 1 2 .0 1 2 .2
2 . 5 .0 0 .0 3 1 .6 1 1 .9 1 2 .0 1 2 .2 1 2 .0
1 2 .0 1 1 .9 1 2 .0
1 3 .9 1 4 .1 1 4 .0
3 . 4 .5 0 .5 2 8 .4 1 4 .0 1 4 .0 1 3 .8 1 4 .0
1 4 .0 1 4 .1 1 3 .9
1 6 .5 1 6 .5 1 6 .3
4 . 4 .0 1 .0 2 5 .3 1 6 .7 1 6 .4 1 6 .4 1 6 .5
1 6 .7 1 6 .6 1 6 .3
2 0 .9 1 9 .5 2 0 .2
5 . 3 .5 1 .5 2 2 .1 2 0 .8 1 9 .8 2 0 .4 2 0 .2
2 0 .9 1 9 .5 2 0 .2
2 5 .2 2 5 .0 2 5 .4
6 *
3 .0 2 .0 1 9 .0 2 5 .2 2 4 .9 2 5 .2 2 5 .2
2 5 .2 2 4 .9 2 5 .2
3 1 .0 3 0 .3 2 9 .6
7 . 2 .5 2 .5 1 5 .8 3 1 .0 3 0 .5 2 9 .8 3 0 .3
3 1 .6 3 0 .2 2 9 .6
TABLE IV (C on tin u ed )
DATA FOR CALIBRATION CURVE
P e rc e n t T r a n s m i s s i o n ^ )
S ta n d a rd
( in m l . )
ls 5 0 H2SO4
( i n m l.)
M icrogram s
o f N itro g e n
Tube Number
S e r i e s 1 2 3 A verage
8 . 2 .0 3 .0 1 2 .6
3 7 .3
3 7 .5
3 7 .6
3 8 .2
3 8 .3
3 8 .3
3 8 .5
3 8 .8
3 8 .5
3 8 .1
9 . 1 .5 3 .5 9 .5
4 8 .0
4 8 .1
4 8 .2
4 9 .2
4 9 .2
4 9 .0
4 9 .8
5 0 .0
5 0 .0
4 9 .1
1 0 . 1 .0 4 .0 6 .3
6 2 .3
6 2 .2
6 2 .2
(C l
oudy)
6 2 .2
1 1 . 0 .5 4 .5 3 .2
7 8 ,9
7 8 .5
7 9 .2
7 8 .1
7 8 .2
7 8 .2
7 8 .0
7 8 .0
7 8 .2
7 8 .4
1 2 . 0 .2 4 .8 1 .3
8 8 .0
8 8 .3
8 8 .5
8 6 .2
8 6 .2
8 6 .5
8 7 .2
8 7 .5
8 6 .8
8 7 .2
^ T h e s ta n d a r d n i tr o g e n s o l u t i o n c o n ta in s 6 ,3 2 m icrogram s o f n i t r o g e n p e r
m i l l i l i t e r .
fr^ P e r c e n t tr a n s m is s io n was d e te rm in e d w ith a Coleman s p e c tr o p h o to m e te r .
23
1 0 0
90
80
70
60
50
C O
c n
S
c n
z
<
a:
i-
40
30
H
Z
UJ
o
20
0 1 0 20 30
MICROGRAMS OF NITROGEN
FIGURE I I I
CALIBRATION CURVE FOR NITROGEN DETERMINATION
24
s o l u t i o n was c a u t i o u s l y added t o th e d i g e s t i o n m ix tu r e .
G e n tle b o i l i n g was c o n tin u e d f o r f i v e m in u te s a f t e r th e
m ix tu re h ad c l e a r e d . F o llo w in g a c o o lin g p e r i o d f o r two
m in u te s* th e s o l u t i o n was d i l u t e d t o 50 m l. w i t h d i s t i l l e d
w a t e r , and was c h i l l e d in an i c e b a t h .
F o r th e NPN d e t e r m i n a t i o n , 1 m l. of serum was t h o r
o u g h ly m ixed w ith 6 m l. o f 10 p e r c e n t t r i c h l o r a c e t i c a c i d
in a c e n t r i f u g e tu b e and c e n t r i f u g e d r a p i d l y . An a l i q u o t
( u s u a l l y 3 t o 5 m l.) o f th e c l e a r s u p e r n a t a n t l i q u i d was
d i g e s t e d w i t h H2SO4 and p o ta s s iu m p e r s u l f a t e , a s d e s c r i b e d
f o r th e t o t a l n i t r o g e n d e t e r m i n a t i o n . The s o l u t i o n was
d i l u t e d t o 25 m l. a f t e r d i g e s t i o n and c h i l l e d .
F o r th e b l a n k , a 1 :5 0 d i l u t i o n o f H2SO4 (C. P .
S p e c i a l — low i n n i t r o g e n ) w i t h w a te r was u s e d .
A n i t r o g e n s t a n d a r d was u se d c o n t a i n i n g 1 9 .0 m ic ro
grams o f n i t r o g e n i n 5 m l. of s o l u t i o n . T h is was p r e p a r e d
b y a d d in g 2 m l. o f t h e 1 :50 H2SO4 t o 3 m l. o f t h e n i t r o g e n
s t a n d a r d w hich c o n ta in e d 6 .3 2 m iorogram s o f n i t r o g e n p e r
m i l l i l i t e r o f s o l u t i o n .
N e s s l e r f s r e a g e n t was p r e p a r e d a c c o r d in g t o t h e f o r
mula o f F o l l n and Wu, d e s c r i b e d b y Wong (1 9 2 3 ).
To 5 .0 m l. o f th e d i g e s t s f o r t o t a l n i t r o g e n and NPN
and t o 5 .0 m l. o f th e b l a n k and th e s t a n d a r d was added 3 .0
m l. of N e s s l e r 's r e a g e n t . The tu b e s were p l a c e d i n th e d a rk
25
f o r t h i r t y m in u te s f o r c o m p lete c o l o r developm ent* The
p e r c e n t t r a n s m i s s i o n was d e te rm in e d w i t h th e Coleman
s p e c tr o p h o to m e te r a t a wave l e n g t h o f 425 m il l im i c r o n s *
C. FIRST EXPERIMENTAL SERIES
1 . P r e l i m i n a r y t r e a t m e n t . Twenty-two C a r r e l f l a s k
c u l t u r e s o f c h ic k e n em bryonic h e a r t t i s s u e were s e t up in
th e way d e s c r i b e d In C h a p te r I I I (page 1 2 ) .
On th e f o llo w in g day, g ro w th of f i b r o b l a s t s was ob
s e rv e d in e a c h f l a s k . A l l f l a s k s were washed f o r t h r e e
f i f t e e n m in u te p e r i o d s w i t h Simms s o l u t i o n s . The w a sh in g
p r o c e s s removed much o f t h e s o l u b l e m a t e r i a l , l e a v in g th e
t i s s u e fra g m e n t in a f i b r i n c l o t .
2 . E x p e rim e n ta l c u l t u r e s . One s e r i e s , e a c h c o n s i s t
in g of f o u r f l a s k s , was u se d f o r e a c h of th e f o u r serum
d i g e s t s . The amount of e a c h d i g e s t and Simms s o l u t i o n w hich
was u s e d i n eac h s e r i e s i s g iv e n i n T ab le V. The volume of
eac h serum d i g e s t which was added was c a l c u l a t e d t o c o n ta in
th e same amount o f n i t r o g e n . Thus eac h c u l t u r e in th e
e x p e rim e n t w i t h th e e x c e p tio n of th e n e g a t iv e c o n t r o l s was
s u p p l i e d w ith th e same amount o f n i t r o g e n . No embryo e x
t r a c t was in c lu d e d in th e s e r i e s o f e x p e r im e n ta l c u l t u r e s .
3 . C o n t r o l c u l t u r e s . T h ree p o s i t i v e c o n t r o l c u l t u r e s
c o n s i s t i n g o f u n d i g e s te d serum and embryo e x t r a c t i n e q u a l
26
TABLE V
AMOUNT OP SERUM DIGEST AND SIMMS
SOLUTION USED IN EACH EXPERIMENTAL CULTURE
C u ltu r e pH o f M i l l i l i t e r s M i l l i l i t e r s o f
Number D ig e s t D ig e s t o f D ig e s t Simms S o l u t i o n
1 - 4 A 2 .0 0 .6 0
O
•
o
5 - 8 B 2 .5 0 .7 1 0 .2 9
9 - 1 2 C 3.0 0 .9 3 0 .0 7
13 - 16 D 3 .5 1 .0 0 0 .0 0
27
volum es w ere In c lu d e d in th e ex p erim en t*
N e g a tiv e c o n t r o l s w ere p r e p a r e d in t h r e e f l a s k s * One
m i l l i l i t e r o f Simms s o l u t i o n was added t o th e t i s s u e f r a g
ment in e a c h o f t h r e e C a r r e l f l a s k s .
4 . S u b seq u en t t r e a tm e n t o f c u l t u r e s . C a r e f u l m ic r o
s c o p ic o b s e r v a t i o n s o f e a c h c u l t u r e were made a t f r e q u e n t
i n t e r v a l s t o e v a l u a t e th e r e l a t i v e r a t e of g ro w th and a l s o
th e h e a l t h and c h a r a c t e r i s t i c s o f th e c e l l s in th e c u l t u r e s .
Im m e d ia te ly a f t e r th e e x p e r im e n ta l and c o n t r o l c u l
t u r e s were s e t up , m ic ro p h o to g ra p h s on 35 mm. f i l m were
ta k e n of th e e n t i r e c u l t u r e in each f l a s k u s i n g a L e ic a
cam era. T h is tim e was c o n s id e r e d z e ro tim e , s i n c e th e
amount of g ro w th in e a c h c u l t u r e was a p p r o x im a te ly t h e same
when th e d i g e s t s were a d d e d . T w e n ty -fo u r and f o r t y - e i g h t
h o u rs l a t e r , more m ic ro p h o to g ra p h s were ta k e n o f e a c h c u l t u r e
t o r e c o r d th e i n c r e a s e in a r e a . P h o to g ra p h s a t h i g h e r
m a g n i f i c a t i o n s were a l s o made t o d e m o n s tra te th e m orphology
and th e changes i n th e s i z e of th e f i b r o b l a s t s a s th e c u l
t u r e s grew .
D. SECOND EXPERIMENTAL SERIES
1. P r e l im i n a r y t r e a t m e n t . The p ro c e d u re in th e
second e x p e r im e n ta l s e r i e s d i f f e r e d somewhat from th e f i r s t
e x p e r im e n t. T w e n ty -th re e f r e s h e x p l a n t s of c h ic k e n em bryonic
h e a r t t i s s u e w ere p la c e d in C a r r e l f l a s k s c o n t a i n i n g 0 .8 m l.
28
o f plasm a p e r f l a s k . The fra g m e n ts w ere c a r e f u l l y c e n te r e d
and l e f t u n d i s t u r b e d f o r one h o u r u n d e r t h e u l t r a - v i o l e t
l i g h t in th e c u l t u r e room. By t h i s m ethod i t was p o s s i b l e
t o o b t a i n e x p la n ts of h e a r t t i s s u e i n plasm a c l o t s w ith o u t
th e a d d i t i o n of any embryo e x t r a c t . The e l i m i n a t i o n o f th e
embryo e x t r a c t i n th e p r e l i m i n a r y t r e a t m e n t o f th e c u l t u r e s
r e s u l t e d in an e x p e rim e n t w hich r e f l e c t e d more a c c u r a t e l y
th e grow th s t i m u l a t i n g f a c t o r s in th e serum d i g e s t s . I t was
q u e s t i o n a b l e w h e th e r th e w ashin g p r o c e s s d e s c r i b e d i n th e
f i r s t e x p e r im e n ta l s e r i e s (page 25) c o m p le te ly removed a l l
th e grow th s t i m u l a t i n g embryo e x t r a c t .
F o r t y - e i g h t h o u r s l a t e r a l l c u l t u r e s were o b se rv e d
u n d e r th e m ic ro sc o p e and a r a t h e r c o n s t a n t amount of grow th
was o b se rv e d in e a c h c u l t u r e . The c u l t u r e s w ere th e n washed
w ith Simms s o l u t i o n a s d e s c r i b e d in th e f i r s t e x p e rim e n t
(page 2 5 ) .
2 . E x p e r im e n ta l c u l t u r e s . S i x t e e n e x p e r im e n ta l c u l
t u r e s were s e t up In th e way d e s c r i b e d on page 25 and in
T ab le V (page 2 6 ) .
3 . C o n tr o l c u l t u r e s . Two p o s i t i v e and two n e g a tiv e
c o n t r o l c u l t u r e s were p r e p a r e d a s d e s c r i b e d In e x p erim e n t
one (page 2 5 ) . In a d d i t i o n , a n e g a tiv e serum c o n t r o l was
u s e d , c o n s i s t i n g o f an e q u a l volume o f u n d i g e s t e d serum and
Simms s o l u t i o n b u t c o n ta in i n g no embryo e x t r a c t .
4 . S u b seq u e n t t r e a t m e n t . A l l c u l t u r e s were p h o to -
29
g ra p h e d im m e d ia te ly f o llo w in g th e a d d i t i o n of th e serum
d i g e s t s . T his was c o n s id e r e d z e ro tim e . At 24 hour i n t e r v a l s
f o r s i x d ay s, a l l c u l t u r e s were p h o to g ra p h e d t o p r o v id e a
r e c o r d o f th e g ro w th of th e c u l t u r e s . In a d d i t i o n , f r e
quent o b s e r v a t i o n s were made o f e a c h c u l t u r e and m ic ro -
p h o to g ra p h s a t h ig h e r m a g n i f i c a t i o n s were made t o p r o v id e
a m ethod of e s t i m a t i n g th e h e a l t h o f th e c e l l s i n th e
c u l t u r e s a s g ro w th p r o g r e s s e d .
At 48 h o u rs and a g a in a t 96 h o u r s , th e c u l t u r e s were
washed tw ic e w i t h Simms s o l u t i o n and f r e s h d i g e s t s w ere
a d d e d . The e x p e rim e n t was t e r m in a t e d a t 144 h o u r s .
CHAPTER V
EXPERIMENTAL DATA
A. SERUM DIGESTS
The r e s u l t s of th e p e p s in d i g e s t i o n o f c h ic k e n serum
a r e re c o r d e d in T able V I. V alu es f o r t o t a l n i t r o g e n and
NPN b e f o r e and a f t e r th e d i g e s t i o n a r e g i v e n .
In T able V I I (page 32) th e d e c r e a s e in t o t a l n i t r o g e n
and NPN a s a r e s u l t of th e en zy m atic h y d r o l y s i s and d i a l y s i s
a r e r e c o r d e d .
B. FIRST EXPERIMENTAL SERIES
I n T able V I I I (pages 33 - 35) a r e r e c o r d e d th e r e s u l t s
o f th e f i r s t e x p e r im e n ta l s e r i e s . Q u a n t i t a t i v e d a t a f o r
g ro w th e x p e rim e n ts i_n v i t r o a r e d i f f i c u l t to o b t a i n and
p r e s e n t . T ab le V I I I c o n ta in s in f o r m a tio n in v e ry g e n e r a l
term s r e g a r d i n g th e r e l a t i v e I n c r e a s e i n a r e a o f th e c u l
t u r e s and th e a p p e a ra n c e and h e a l t h of th e c e l l s . The
gro w th of e x p e r im e n ta l c u l t u r e s in th e d i g e s t s p r e p a r e d a t
pH 2 .0 and 3 .0 a p p r o x im a te ly e q u a le d th e g ro w th In th e
p o s i t i v e c o n t r o l c i i l t u r e s .
The g ro w th o f th e p o s i t i v e c o n t r o l c u l t u r e s was en
t i r e l y norm al w ith th e u s u a l p r o l i f e r a t i o n o f f i b r o b l a s t s .
N e g a tiv e c o n t r o l c u l t u r e s showed a s m a ll amount of
TABLE VI
31
RESULTS OP PEPSIN DIGESTION OF SERUM PROTEINS
D ig e s t
pH o f
D ig e s t
T o ta l N itr o g e n ^ a )
B e fo re A f t e r
D i g e s t io n D i g e s t io n
NPN^a ^
B efo re A f t e r
D i g e s t io n D i g e s t i o n
A 2 .0 6 .8 0 5 .7 0 0 .2 2 0 0 .1 2 0
B 2 .5 6 .7 1 4 .7 3 0 .2 5 7 0 .1 4 0
C 3 .0 5 .3 8 3.68 0 .1 8 3 0 .0 6 3
D 3 .5 5 .2 5 3 .4 3 0 .2 5 0 0 .0 8 0
^a ^In m il l ig r a m s p e r m i l l i l i t e r .
32
TABLE V II
DEGREASE IN TOTAL NITROGEN AND NPN
DURING DIGESTION AND DIALYSIS
D ig e s t
pH o f
D ig e s t T o t a l N itro g e n ^ a ) NPN^a )
A 2 .0 1.10 0 .1 0 0
B 2 .5 1.98 0 .1 1 7
C 3 .0 1 .7 0 0 .1 2 0
D 3 .5 1.82 0 .1 7 0
fa}
' ' I n m il l ig r a m s p e r m i l l i l i t e r .
TABLE V III
RESULTS OP THE FIRST EXPERIMENTAL SERIES
C u ltu re
Number
pH of
D ig est
Time
in Hours
In c re a s e
in Area
C h a r a c t e r i s t i c s
of F i b r o b l a s t s
_ _
M ig ra tio n Comments
1 - 4 2 .0 24 Moderate i n
c re a s e in
a re a
Rounded c e l l s
w ith f a t t y
v a c u o le s in
many c e l l s ;
a few s p in d le
shaped c e lls .
Not much mi
g r a t i o n ; a
sm all, dense
a re a of
grow th d i r
e c t l y around
e x p la n t
pH and c lo t
a re norm al;
growth and
h e a lth of
f i b r o b l a s t s
not q u i te as
good as in
p o s i t i v e
c o n tr o l
c u l t u r e s
1 - 4 2 .0 48
Area d e f i
n i t e l y l a r
g e r th an a t
24 hours
Many t o x i c
c e l l s and
few new f i b r o
b l a s t s
Not much
change
sin c e 24
hours
These c u l
t u r e s appear
t o have
rea ch e d
maximum
grow th and
a r e v e ry
to x ic
03
03
TABLE V I II (co n tin u ed )
RESULTS OF THE FIRST EXPERIMENTAL SERIES
C u ltu re
Number
pH of
D ig e st
Time
in Hours
In c re a s e
in Area
C h a r a c t e r i s t i c s
of F i b r o b l a s t s M ig ratio n Comments
5 - 8 2 .5 24 Not as
much as
in th e
c u ltu r e s
1 - 4
Many h e a l th y
c e l l s ; a few
w ith f a t t y
v a cu o les
Some mi
g r a t i o n ;
grow th
not as
dense as
c u lt u r e s
1 - 4
pH and c l o t
a re norm al;
growth not
as good as
in c u l t u r e s
1 - 4
5 - 8 2.5 48 Same as
a t 24
hours
Many to x ic
c e l l s ; much
d e g e n e ra tio n
of c e l l s ;
few new c e l l s
Same as
a t 24
hours
Same as a t
24 hours
9
(and)
10
3 .0
24
(and)
48
The c l o t s were f l o a t i n g in th e s e two c u lt u r e s soon
a f t e r th e d i g e s t was added. No grow th was o b se rv ed .
TABLE V III (co n tin u ed )
RESULTS OP THE FIRST EXPERIMENTAL SERIES
C u ltu re
Humber
pH of
D ig e st
Time in
Hours
In c re a s e
in Area
C h a r a c t e r i s t i c s
of F i b r o b l a s t s M ig ra tio n Comments
11
(and)
12
3 .0 24 D e f in i t e in
c re a s e in
a re a c o r r e
sponding to
p o s i t i v e
c o n tr o l c u l
t u r e s
Pine network
of h e a lth y
c e l l s ; many
normal f i b r o
b l a s t s ; few
to x ic c e l l s ;
resem ble p o s i
t i v e c o n tro ls
Some m ig ra
tio n ; main
a r e a i s v e ry
dense and
f i l l e d w ith
f i b r o b l a s t s
These c u l
t u r e s have
l a r g e s t
a r e a of
grow th;
most h e a l tl y
c e l l s ; pH
and c l o t
normal
11
(and)
12
3 .0 48 Area of
c u lt u r e has
in c re a s e d
s in c e 24
hours
Many h e a lth y
f i b r o b l a s t s ;
numerous to x ic
c e l l s ; some
a re d i s i n t e
g r a te d
Dense a re a
of grow th;
l i t t l e mi
g r a t i o n
Same as a t
24 hours
13 - 16 3 .5 24 S m a lle st
in c r e a s e in
a re a of any
of th e c u l
t u r e s
Rounded c e l l s ;
f a t t y v a cu o les
in many c e l l s ;
many d i s i n t e
g r a te d c e l l s
Very l i t t l e
m ig ra tio n ;
a very
sm all dense
a re a of new
c e l l s
pH s l i g h t l y
a c id ; p o o r
e s t of a l l
e x p e rim e n ta l
c u lt u r e s
13 - 16 3 .5 48
P r a c t i c a l l y no change in any of th e c h a r a c t e r i s t i c s
of th e c u lt u r e s sin c e th e 24 hour p e r io d .
36
g ro w th , h e a l t h y f i b r o b l a s t s and much c e l l u l a r m i g r a t i o n .
0 . SECOND EXPERIMENTAL SERIES
D ata f o r th e second e x p e r im e n ta l s e r i e s were o b ta in e d
in a d i f f e r e n t way th a n f o r th e f i r s t e x p e r i m e n t a l s e r i e s .
P la n im e te r m easurem ents were made o f th e a r e a o f each
o f th e c u l t u r e s in e a c h d i g e s t a t e a c h 24 h ou r i n t e r v a l .
These m easurem ents were o b ta in e d from e n la rg e m e n ts o f th e
m ic ro p h o to g ra p h s ta k e n o f e a c h c u l t u r e and a r e r e c o r d e d in
T ab le IX. A verages o f th e p l a n i m e t e r r e a d i n g s o f c u l t u r e s
in e a c h d i g e s t a r e g iv e n i n T a b le X (page 3 8 ).
V alu es f o r th e p e r c e n t i n c r e a s e i n a r e a a r e p r e s e n t
ed i n T ab le XI (page 3 8 ) . These v a lu e s were c a l c u l a t e d a c
c o rd in g t o th e e q u a t i o n :
A = At - A0 x 1Q0
Ao
where A s P e r c e n t i n c r e a s e in a r e a
A q s A rea o f c u l t u r e a t z e ro tim e
At = A rea of c u l t u r e a t th e s p e c i f i c tim e
t h a t A i s c a l c u l a t e d .
Curves r e p r e s e n t i n g th e p e r c e n t i n c r e a s e in a r e a of
c u l t u r e s grown in e ac h d i g e s t a r e shown in F ig u r e IV (page
3 9 ) .
M ic ro p h o to g ra p h s a t 24 h o u r i n t e r v a l s of t y p i c a l
c u l t u r e s grown i n e a c h d i g e s t a r e re p ro d u c e d i n F ig u r e V
a , b , c, and d (pages 40 - 4 3 ) .
TABLE IX
37
PLANIMETER MEASUREMENTS 0? CULTURES
IN THE SECOND EXPERIMENTAL SERIES
iu lt u r e
fumber
pH of
D ig e s t 0 24 48 72 96 120 144
1.
2 .0 56 146 192 231 251 234 250
2 .
ft
39 84 137 164 197
------
5. 2 .5 42 79 143 165 160
6.
it
47 70
------ ------ ------
7 .
tt
43 74
------ ------ ------
8 .
tt
26 45 100 142
------
9. 3 .0 68 94 101 209 213 228 261
10.
tt
56 92 127
------ ------ ------ ------
11.
it
53 73 97 206 205 256 281
12.
it
50 82 112 ------
------ ------ ------
13. 3 .5 53 53 50 146 122
14.
it
48 65 79 168 141
15.
ti
55 67 107
TABLE X
38
AVERAGE PLANIMETER READINGS OP CULTURES IN
EACH DIGEST OP THE SECOND EXPERIMENTAL SERIES
pH of
D ig e s t
Time in Hours
D ig e s t 0 24 48 72 96 120 144
A 2 .0 48 115 170 198 224 234 250
B 2 .5 40 67 121 154 160
----- -----
C 3 .0 57 85 109 208 209 242 271
D 3 .5 52 61 65 140 132
...... _ _ _
TABLE XI
PER CENT INCREASE IN AREA OP CULTURES
IN THE SECOND EXPERIMENTAL SERIES
D ig e s t
pH o f
D ig e s t
Time in Hours
0 24 48 72 96 120 144
A 2 .0 0 139 244 312 367 388 422
B 2 .5 0 67 200 295 300
----- -----
C 3 .0 0 50 91 265 265 325 375
D 3 .5 0 18 25 168 154
“ — —
- - -
39
400
<
UJ 300
C E
<
LU
c n
<
LlI
CE
o
z
200
H -
z
LU
O
CE
LU
a.
100
96 48 72 24 44
TIME IN HOURS
FIGURE IV
CURVES SHOWING PER CENT
INCREASE IN AREA OF CULTURES GROWN IN SERUM DIGESTS:
A (pH 2 , 0 ) , B (pH 2 . 5 ) , C (pH 3 .0 ) and D (pH 3 .5 )
TIM E
IN
HOURS
FIGURE V (a)
MICROPHOTOGRAPHS OF CULTURES IN DIGESTS:
A (pH 2 .0 ) and B (pH 2 .5 ) FROM 0 TO 72 HOURS
TIME
IN
HOURS
FIGURE V (b)
MICROPHOTOGRAPHS OF CULTURES IN DIGESTS:
A (pH 2 .0 ) and B (pH 2 .5 ) FROM 96 TO 144 HOURS
42
TIM E
IN
HOURS
FIGURE V (c)
MICROPHOTOGRAPHS OF CULTURES IN DIGESTS:
C (pH 3 .0 ) and D (pH 3 .5 ) FROM 0 TO 72 HOURS
FIGURE V (d)
MICROPHOTOGRAPHS OF CULTURES IN DIGESTS:
(pH 3 .0 ) and D (pH 3 .5 ) FROM 96 TO 144 HOURS
44
The u s u a l m ic r o s c o p ic o b s e r v a t i o n s w ere a l s o made i n
t h e second e x p e r im e n ta l s e r i e s . R e s u l t s o f th e s e o b s e r v a
t i o n s f o r th e second s e r i e s w ere s i m i l a r t o th o s e i n th e
f i r s t s e r i e s r e c o r d e d in T a b le V I I I (p ag es 33 - 3 5 ) .
The c u l t u r e s g row ing i n d i g e s t s p r e p a r e d a t pH 2 .0
and 3 .0 a p p e a re d t o have th e h e a l t h i e s t g row th and th e
g r e a t e s t p r o l i f e r a t i o n o f f i b r o b l a s t s , and were q u i t e s i m i
l a r t o th e p o s i t i v e c o n t r o l c u l t u r e s . Growth o f c u l t u r e s i n
th e d i g e s t p r e p a r e d a t pH 2 .5 a l s o a p p e a re d t o be good b u t
was a l i t t l e l e s s den se th a n g ro w th i n t h e d i g e s t s p r e p a r e d
a t pH 2 .0 and 3 . 0 .
The d i g e s t p r e p a r e d a t pH 3 .5 evoked th e l e a s t g ro w th
r e s p o n s e and th e g ro w th was much p o o r e r th a n grow th in th e
p o s i t i v e c o n t r o l c u l t u r e s . In f a c t , th e g row th in th e d i
g e s t p r e p a r e d a t pH 3 .5 was o n ly s l i g h t l y b e t t e r th a n th e
n e g a t iv e c o n t r o l .
The p o s i t i v e c o n t r o l s showed norm al grow th and th e
n e g a tiv e serum c o n t r o l s a l s o showed a s m a ll amount of grow th
w hich was b e t t e r th a n th e grow th o b se rv e d in th e n e g a tiv e
c o n t r o l in Simms s o l u t i o n a l o n e . T h ere was much m i g r a t i o n
In th e n e g a t iv e Simms c o n t r o l b u t l i t t l e g ro w th .
A d d itio n o f f r e s h d i g e s t s a f t e r w ashin g w i t h Simms a t
48 h o u rs p ro d u ce d a m arked re s p o n se * A d e f i n i t e improvement
in th e h e a l t h o f th e c e l l s was o b se rv e d a t 72 h o u r s , one day
f o l lo w in g f r e s h a d d i t i o n of th e d i g e s t .
45
A s i m i l a r im provem ent was o b se rv e d in two o f th e e x
p e r i m e n t a l c u l t u r e s when f r e s h d i g e s t s were added a t 96
h o u r s . A p p r e c ia b le i n c r e a s e s i n th e s u r f a c e a r e a of th e
c u l t u r e s were n o te d 24 h o u rs a f t e r th e a d d i t i o n o f f r e s h
d i g e s t s . These c hanges can be se e n i n F ig u r e IV (page 3 9 ).
The c u l t u r e s grow ing i n th e d i g e s t s p r e p a r e d a t pH
3 .5 and 2 .5 d ie d b e f o r e 96 h o u r s . C u l tu r e s in th e o t h e r two
d i g e s t s c o n tin u e d t o p r o l i f e r a t e , p r o d u c in g many h e a l t h y
c e l l s u n t i l 144 h o u rs when th e e x p e rim e n t was t e r m i n a t e d .
CHAPTER VI
DISCUSSION OP RESULTS
A. EFFECTS OF PEPSIN ON SERUM PROTEINS
A c o n s i d e r a t i o n o f th e r e s u l t s i n T a b le V II (page 32)
does n o t le a d t o any w e l l d e f in e d c o r r e l a t i o n betw een th e
pH a t w hich th e d i g e s t i o n was p r e p a r e d and th e d e c r e a s e in
th e t o t a l n i t r o g e n and NPN d u r in g d i g e s t i o n and d i a l y s i s .
The d e c r e a s e in t o t a l n i t r o g e n was r a t h e r c o n s t a n t
f o r th e d i g e s t i o n s a t pH 2 .5 , 3 .0 and 3 . 5 . The d e c r e a s e In
t o t a l n i t r o g e n in th e d i g e s t a t pH 2 .0 was much l e s s th a n
in th e o t h e r t h r e e d i g e s t s . A com parison o f th e d i g e s t s a t
pH 2 .0 and 2 .5 shows t h a t th e d e c r e a s e i n t o t a l n i t r o g e n a t
2 .0 was o n ly a b o u t 50 p e r c e n t o f t h a t a t pH 2 . 5 , T his f a c t
can b e c o r r e l a t e d w i t h th e e x p e r im e n ta l r e s u l t s in term s o f
r e l a t i v e g ro w th w hich w i l l be d i s c u s s e d l a t e r in t h i s c h a p te r.
The v a lu e s o b ta in e d f o r th e l o s s o f NPN d u r in g th e
d i g e s t i o n and d i a l y s i s w ere s u r p r i s i n g f o r i t had b e e n a n
t i c i p a t e d t h a t th e amounts o f NPN re m a in in g i n th e d i g e s t
f o l lo w in g th e d i a l y s i s p e r i o d would b e n e g l i g i b l e .
I t was c a l c u l a t e d t h a t any d i a l y z a b l e m a t e r i a l would
have b een d i l u t e d by a f a c t o r o f 400. NPN In su c h sm a ll
am ounts would n o t have been d e t e c t e d by th e method of a n a l y
s i s u se d in t h i s e x p e r im e n t.
47
However NPN was p r e s e n t in q u a n t i t i e s w hich were
e a s i l y d e t e c t a b l e a f t e r th e d i g e s t i o n and d i a l y s i s . The
d e c r e a s e in NPN v a r i e d d i r e c t l y w i t h th e pH of th e d i g e s
t i o n w i t h t h e g r e a t e s t l o s s of NPN in th e d i g e s t i o n a t pH
3 .5 and th e s m a l l e s t l o s s o f NPN in th e d i g e s t i o n a t pH 2 . 0 .
These r e s u l t s s u g g e s t th e p o s s i b i l i t y t h a t a low
m o le c u la r w e ig h t p o l y p e p t i d e was s p l i t from th e serum p r o
t e i n s b y th e a c t i o n o f p e p s i n . T h is p o l y p e p t i d e was l a r g e
enough t o be in c o m p le te ly d i a l y z e d d u r in g th e d i a l y s i s
w hich to o k p l a c e f o r a p e r i o d o f 144 h o u r s . T h is p o ly p e p
t i d e had a low m o le c u la r w e ig h t and was s o l u b l e in 10 p e r
c e n t t r i c h l o r a c e t i c a c i d .
T his e x p l a n a t i o n c o u ld a c c o u n t f o r th e d e c r e a s e i n
t o t a l n i t r o g e n d u r in g th e d i g e s t i o n w ith o u t a com p lete
rem o v a l o f NPN b y d i a l y s i s .
B. EFFECT OF DIGESTS ON GROWTH OF CULTURES
In b o t h e x p e r im e n ta l s e r i e s i t was e v id e n t t h a t th e
f o u r d i g e s t s w ere c a p a b le o f s u p p o r t i n g th e gro w th o f t i s s u e
c e l l s i n v i t r o f o r s h o r t p e r i o d s o f tim e . In th e second
e x p e r im e n ta l s e r i e s , g ro w th o f t i s s u e c e l l s _in v i t r o was
s u s t a i n e d f o r o v e r s i x days i n th e d i g e s t s a t pH 2 .0 and
3 .0 . I t i s p o s s i b l e t h a t th e d e a t h o f th e c u l t u r e s a f t e r
t h r e e day s in th e d i g e s t s p r e p a r e d a t pH 2 .5 and 3 .5 was
48
due t o f a c t o r s o t h e r t h a n th e d i g e s t s . The f a c t t h a t grow th
o c c u r r e d f o r t h r e e d ay s in a l l d i g e s t s , and f o r s i x days in
two of th e d i g e s t s i s s i g n i f i c a n t e v id e n c e f o r th e p r e s e n c e
of g ro w th s t i m u l a t i n g f a c t o r s i n th e p e p s in d i g e s t e d homo
lo g o u s serum .
The c u rv e s in F ig u r e IV (page 39) show t h a t a d e f i n i t e
c o r r e l a t i o n e x i s t s "between th e pH a t w hich th e d i g e s t s were
p r e p a r e d and th e p e r c e n t i n c r e a s e in a r e a o f th e c u l t u r e s .
The g r e a t e s t i n c r e a s e in a r e a was o b se rv e d i n c u l t u r e s grown
in th e d i g e s t p r e p a r e d a t pH 2 . 0 . As th e pH a t w h ich th e
d i g e s t s w ere p r e p a r e d i n c r e a s e d , t h e p e r c e n t I n c r e a s e in
a r e a of th e c u l t u r e s grown i n th e d i g e s t s d e c r e a s e d .
F ig u r e IV (page 39) a l s o i l l u s t r a t e s th e e f f e c t s o f
f r e s h a d d i t i o n o f th e d i g e s t s t o th e c u l t u r e s . The a r e a o f
c u l t u r e s grow ing in d i g e s t s p r e p a r e d a t pH 5 . 5 and 3 .0 showed
a marked i n c r e a s e a t 72 h o u r s , one day f o l lo w in g th e a d d i
t i o n o f f r e s h d i g e s t . T h is e f f e c t was r e p e a t e d when f r e s h
d i g e s t was added a t 96 h o u rs t o c u l t u r e s grow ing i n th e d i
g e s t p r e p a r e d a t pH 3 . 0 .
C u l t u r e s in d i g e s t s a t pH 2 .5 and 2 .0 showed a more
u n ifo rm i n c r e a s e In a r e a w i t h no sudden i n c r e a s e s when f r e s h
d i g e s t s w ere added t o th e c u l t u r e s a t 48 and 96 h o u r s .
The i n c r e a s e in a r e a o f c u l t u r e s i s n o t a t r u e
c r i t e r i a of a c t u a l grow th s i n c e m i g r a t i o n of c e l l s a s w e l l
49
a s new g row th o f c e l l s w i l l cause an i n c r e a s e in a r e a of th e
t i s s u e c u l t u r e s . I'hese d a t a r e g a r d i n g th e p e r c e n t i n c r e a s e
in a r e a o f th e t i s s u e c u l t u r e s must he i n t e r p r e t e d w ith
c a u t i o n , how ever, a s P a rk e r (1958) and F i s c h e r (1946) have
i n d i c a t e d , s i g n i f i c a n t d i f f e r e n c e s su ch as th o s e p r e s e n t e d
i n F ig u r e IV (page 39) can he i n t e r p r e t e d in term s o f g ro w th
s in c e th e d i f f e r e n c e s in th e a r e a o f c u l t u r e s a t th e v a r i o u s
tim e s were g r e a t enough so t h a t m i g r a t i o n a lo n e c o u ld n o t
a c c o u n t f o r a l l o f th e v a r i a t i o n .
The o b s e r v a t i o n s i n d i c a t e t h a t c le a v a g e p r o d u c ts o f
p r o t e i n s o b t a i n e d b y p e p s i n d i g e s t i o n n e a r e r t h e pH optimum
f o r th e enzyme, p ro d u ce d s u b s ta n c e s t h a t w ere u t i l i z e d a t a
more u n ifo rm r a t e by th e t i s s u e c e l l s in_ v i t r o f o r th e
s y n t h e s i s o f new p r o t e i n .
P e p s in d i g e s t i o n of serum p r o t e i n s a t pH 2 .5 , 3 .0 ,
and 3 .5 p ro d u ced s u b s ta n c e s , p a r t o f w h ich w ere r a p i d l y
u t i l i z e d by th e t i s s u e c e l l s f o r p r o t e i n s y n t h e s i s . The a c
t i v i t y o f t h e s e s u b s ta n c e s was r e f l e c t e d by th e sudden i n
c r e a s e in a r e a o f th e c u l t u r e s l e s s th a n 24 h o u rs f o llo w in g
th e a d d i t i o n o f th e d i g e s t s a t th e 48 h o u r p e r i o d .
I n c o n c lu s io n , th e r e s u l t s o f th e two e x p e rim e n ts
p r e s e n t e d c o n firm ed p a r t o f th e work o f F i s c h e r (1 9 4 7 ). In
a d d i t i o n , th e r e s u l t s i n d i c a t e d t h a t a g ra d e d h y d r o l y s i s
o f serum p r o t e i n s w i t h p e p s in was a c c o m p lish e d b y v a r y in g
50
th e pH a t w hich th e d i g e s t i o n was p re p a re d * Amounts of non-
d i a l y z a b l e s u b s ta n c e s w hich v a r i e d w i t h th e pH o f th e d i g e s t
w ere p ro d u c e d . These s u b s ta n c e s p o s s e s s e d a v a r i a b l e g ro w th
s t i m u l a t i n g a c t i v i t y f o r t i s s u e c e l l s in v i t r o * E vid en ce
f o r t h i s v a r i a t i o n was seen i n th e d i f f e r e n c e s in th e r a t e s
and d e g re e o f grow th o f th e c u l t u r e s in th e f o u r serum d i
g e s t s *
The b e s t grow th o c c u r r e d i n th e serum d i g e s t p r e p a r e d
a t pH 2 . 0 . At t h i s pH, th e d e c r e a s e in th e t o t a l n i t r o g e n
c o n te n t was c o n s i d e r a b ly l e s s th a n in any o f th e o t h e r d i
g e s t s . T h is f a c t s u g g e s te d t h a t p o s s i b l y th e q u a n t i t y o f
p r o t e i n w hich was h y d ro ly z e d b y th e p e p s in a t pH 2 .0 was
s p l i t t o p ro d u ce a g r e a t e r amount o f th e grow th p ro m o tin g
s u b s t a n c e . T h is s u b s ta n c e had th e c o r r e c t m o le c u la r w e ig h t
and c o n f i g u r a t i o n and was d i r e c t l y s y n t h e s i z e d i n t o new
p r o t e i n by th e t i s s u e c e l l s grov/ing in v i t r o .
CHAPTER V II
SUMMARY
The g row th s t i m u l a t i n g e f f e c t s o f th e n o n - d i a l y z a b l e
p r o d u c t s o f p e p s i n d i g e s t e d hom ologous serum p r o t e i n s f o r
t i s s u e c e l l s c u l t i v a t e d i n v i t r o have b e en s t u d i e d in t h i s
i n v e s t i g a t i o n .
S im u lta n e o u s d i g e s t i o n and d i a l y s i s of c h ic k e n serum
p r o t e i n s were c a r r i e d o u t a t a c o n s t a n t te m p e r a tu r e in
b u f f e r s in w hich th e pH v a r i e d by 0*5 o f a pH u n i t from
pH 2 .0 t o 3 .5 .
A d d itio n of t h e s e d i g e s t s t o C a r r e l f l a s k c u l t u r e s of
c h ic k e n em bryonic h e a r t t i s s u e i n c h ic k e n plasm a c l o t s p r e
p a re d w ith no embryo e x t r a c t r e s u l t e d in t h e g ro w th o f
t i s s u e c e l l s In e v e ry d i g e s t .
The g ro w th r e s p o n s e was g ra d e d w ith a maximum grow th
e f f e c t in th e d i g e s t p r e p a r e d a t pH 2 . 0 . T h is e f f e c t was
com parable t o t h a t o b s e rv e d i n norm al t i s s u e c u l t u r e s h a v in g
embryo e x t r a c t a s th e g row th f a c t o r .
I t was co n clu d ed t h a t th e n o n - d i a l y z a b l e p r o d u c ts o f
p e p s in d i g e s t e d hom ologous serum p r o t e i n s were g ro w th s tim
u l a t i n g f o r t i s s u e c e l l s grow ing i n v i t r o and can be u sed
by th e c e l l s f o r th e s y n t h e s i s o f new p r o t e i n . The d e g re e
o f g ro w th s t i m u l a t i o n v a r i e d a c c o rd in g t o t h e pH a t w hich
th e p r o t e i n d i g e s t was p r e p a r e d .
BIBLIOGRAPHY
A s tr u p , T. and A. F i s c h e r , "V I. P r o t e i n M etab o lism o f
T is s u e C e l l s i n v i t r o . ” A cta P h y s i o l . S c a n d .,
14: 195, 1947.
B aker, L. e . and A. C a r r e l , " E f f e c t o f Amino A cids and
D ia ly z a b l e C o n s t i t u e n t s o f Em bryonic T is s u e J u i c e on
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Creator Williams, Albert Doran (author)

Core Title The effects of the non-dialyzable products of pepsin digested serum proteins on the growth of tissue cells in vitro

School Department of Biochemistry and Nutrition

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1949-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776468

Unique identifier UC11348844

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Legacy Identifier EP41299.pdf

Dmrecord 776468

Document Type Thesis

Rights Williams, Albert Doran

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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